Several electrophysiology techniques have been used for many years to produce high quality data. Most cellular electrophysiology techniques are either patch clamp or sharp electrode. Patch clamp traditionally uses a glass micropipette, with an open tip diameter of about 1-2 μm. The tip is formed into a smooth surfaced circle. The patch clamp electrode tip is pressed against the cell membrane and suction is applied causing the cell to form a tight seal or “gigaohm seal” with the electrode. This is contrasted by the sharp electrode methods where the electrode is used to impale the cells to conduct traditional intracellular recordings.
The interior of the pipettes are filled with different solutions depending on the specific ion measurements or techniques used. For example, with whole cell recordings, a solution that approximates the intracellular fluids is used. A metal electrode in contact with this solution conducts the electrical signals to an amplifier. The researcher can change the composition of this solution or add drugs to study the ion channels under different conditions. The patch clamp technique was developed in the late 1970s and early 1980s.
Electrophysiology techniques are technically demanding and typically require highly trained operators. The resulting data is of extremely high fidelity; however, this also means that external electrical noises need to be diligently managed. Lighting, electronics, grounding, connections, building wiring, and radio waves can all introduce noise into the system. The electrophysiology electronics and equipment are considerably expensive and technically demanding to assemble and maintain. The micropipettes used for patch clamp are also a point of consideration for the researcher. Typically the researcher fabricates the micropipette and forges the tip at or about the time it is to be used. Additionally, the pipettes need to be carefully filled with the pipette solution. All in all, existing electrophysiology techniques are highly complex and often prohibitively expensive.
A need exists for an improved cellular physiology technique that has lower barriers to entry and is less technically demanding than electrophysiology.
Over the last several years physiologically active dyes have been developed and successfully commercialized to partially address the technical and practical limitations of existing electrophysiology techniques. These dyes are designed to be sensitive to the concentration of specific ion species (e.g. Ca++, Na+, K+). Some of these dyes are sensitive to electrical potential changes.
Most physiologically active dyes are imaged by a technique called ratiometric imaging. Ratiometric imaging relies on the variable excitation properties of the dyes. The dyes preferentially excite at different wavelengths, depending on the concentration of the specific ion or potential. The emission spectrum of the dyes, however, remains the same. By alternating between the excitation wavelengths and correlating the relative emission outputs, the ionic concentrations or electrical potential can be determined and the changes can be plotted over time.
Modern ratiometric illumination systems are capable of alternating at up to 250 cycles/second. Techniques have also been developed that use baseline excitation/emission ratios to calculate the physiologic changes. By comparing the baseline with continuous single wavelength excitation imaging, very high sampling rates can be realized.
The majority of ratiometric imaging systems are constructed around a specialized light source that alternates the excitation energy, a fluorescent microscope, and an intensified CCD video camera system. Alternatively, a confocal microscope system can be configured to do ratiometric imaging. These systems are designed to illuminate the entire cell and capture the resulting images. This becomes problematic over extended imaging periods since the fluorescent dyes are progressively photobleached and the emission strength decreases. Maintaining consistent emission ratios is critical for conducting these techniques. Likewise, once the dyes have been sufficiently bleached, no further data can be gathered.
Needs exist for an improved physiological active dye imaging technique that does not expose the sample with widefield, whole sample, or propagating excitation energy.
Total internal reflection fluorescence microscopy (TIRF) is an technique that allows the researcher to excite a very thin layer of the sample that is in close (˜100 nm) proximity to the coverslip or microscope slide. TIRF is accomplished by introduce light into the slide or coverslip at an incident angle that causes the light to be reflected internally. This internal reflection generates an evanescent field on the surface to the glass. The evanescent field will excite appropriate fluorescent molecules if they are within field. As the distance from the glass surface increases, the evanescent field intensity falls exponentially. The practical application of TIRF microscopy provides for a single plane of imaging that is not obscured by objects above and below the focal plane.
Needs exist for user directed, point specific TIRF techniques.